CN102959084A - 有用物质的制备方法 - Google Patents
有用物质的制备方法 Download PDFInfo
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- CN102959084A CN102959084A CN2011800321416A CN201180032141A CN102959084A CN 102959084 A CN102959084 A CN 102959084A CN 2011800321416 A CN2011800321416 A CN 2011800321416A CN 201180032141 A CN201180032141 A CN 201180032141A CN 102959084 A CN102959084 A CN 102959084A
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Abstract
本发明提供涉及利用微生物的有用物质的制备方法，缩短微生物的培养期间，改善有用物质的生产性的方法。通过作为主培养初始培养基使用碳水化物和烃的氧化物的合计浓度以碳当量计不足0.4重量%的，升高微生物消费碳水化物和/或烃的氧化物的速度，升高目的有用物质的生产性。
Description
【技术领域】
本发明涉及利用微生物的有用物质的制备方法。
【背景技术】
作为利用微生物的有用物质的制备方法，已知由发酵法的L-氨基酸、核酸、不饱和脂肪酸的制备等（非专利文献1、2）。这些有用物质以葡萄糖等的碳水化物和/或烃的氧化物作为底物，利用微生物的代谢制备出。作为用于制备有用物质的微生物的培养方法，已知使初始培养基含底物，添加全部的培养基成分而培养的回分培养、向接种微生物的初始培养基添加一部分的底物而开始培养之后，将其余的底物间歇地或连续地培添加到养基中培养的流加培养、向发酵槽以一定速度供给含底物的新鲜培养基，将这和同量的培养液连续地排出槽外而进行培养的连续培养等。但是，在任何的培养方法中，在工业地制备有用物质的生产培养基中，在接种微生物的时间点，含成为有用物质的底物的葡萄糖、淀粉糖化物等的碳水化物和/或烃的氧化物。还不知不向初始培养基添加葡萄糖、淀粉糖化物等的碳水化物和/或烃的氧化物而开始微生物的培养的方法。
【现有技术文献】
【非专利文献】
非专利文献1：应用微生物学改订版、培风馆、1993年发行
非专利文献2：科学和工业、第74卷1号、26（2000）
【发明内容】
【发明要解决的技术课题】
本发明涉及利用微生物的有效制备目的有用物质的方法，更具体而言以提供缩短有用物质生产中要求的培养期间，提高有用物质的生产效率的方法作为目的。
【解决课题的技术方案】
本发明人为了解决上述课题重复了锐意检查，发现在由微生物培养制备有用物质的方法中，作为主培养初始培养基，使用碳水化物和烃的氧化物的合计浓度以碳当量计不足0.4重量%，通过在微生物开始对数增殖之后，添加碳水化物和/或烃的氧化物有效制备有用物质是可能的，从而完成本发明。
即，本发明包括以下的发明，但不限于此。
（1）由微生物培养制备有用物质的方法，其包括：微生物的预培养步骤、将得到的预培养液接种到主培养初始培养基的步骤，其中在主培养初始培养基中的碳水化物和烃的氧化物的合计浓度以碳当量计不足0.4重量%，及微生物开始对数增殖之后，添加碳水化物和/或烃的氧化物的主培养步骤。
（2）（1）所述的制备方法，其中主培养初始培养基实质上未添加碳水化物和/或烃的氧化物。
（3）（1）或（2）所述的制备方法，其中微生物开始对数增殖之后添加的碳水化物和/或烃的氧化物是糖类。
（4）（1）～（3）之任一项所述的制备方法，微生物是将碳水化物和/或烃的氧化物由外型淀粉酶异化的微生物。
（5）（1）～（4）之任一项所述的制备方法，其中微生物是被孢霉（Mortierella）属微生物。
（6）（1）～（5）之任一项所述的制备方法，其中有用物质是高度不饱和脂肪酸。
【发明效果】
由本发明，可有效进行由微生物的有用物质的生产。
【附图说明】
【图1】图1是示在向实施例1的初始培养基添加葡萄糖的培养系统、及未向初始培养基添加葡萄糖的培养系统中，在(5Z,8Z,11Z,14Z)-5,8,11,14-二十碳四烯酸的生产实验的培养基中的葡萄糖浓度的推移的图。
图2是示在向实施例1的初始培养基添加葡萄糖的培养系统、及未向初始培养基添加葡萄糖的培养系统中，在(5Z,8Z,11Z,14Z)-5,8,11,14-二十碳四烯酸的生产实验的总脂肪酸中所占的(5Z,8Z,11Z,14Z)-5,8,11,14-二十碳四烯酸的比例的推移的图。
【实施方式】
如上所述，本发明是由微生物培养制备有用物质的方法，其包括：微生物的预培养步骤、将得到的预培养液接种到主培养初始培养基的步骤，其中在主培养初始培养基中的碳水化物和烃的氧化物的合计浓度以碳当量计不足0.4重量%，及微生物开始对数增殖之后，添加碳水化物和/或烃的氧化物的主培养步骤。
由本发明制备的有用物质只要是可由微生物生产的物质，就不特别限定，可举出氨基酸、核酸、不饱和脂肪酸、脂质、蛋白质、肽、维生素、糖、糖醇、醇、有机酸、抗生物质、生理活性物质等。
作为氨基酸，可举出L-谷氨酸、L-赖氨酸、L-苏氨酸、L-色氨酸、L-苯丙氨酸、L-酪氨酸、L-亮氨酸、L-异亮氨酸、L-缬氨酸、L-天冬酰胺、L-天冬氨酸、L-组氨酸、L-谷氨酰胺、L-精氨酸、L-鸟氨酸、L-瓜氨酸、L-脯氨酸、L-丝氨酸、甲硫氨酸、L-丙氨酸、L-半胱氨酸、胱氨酸、高丝氨酸等，作为核酸，可举出肌苷、鸟苷、肌苷酸、鸟苷酸、黄苷酸、胞苷酸等。
作为不饱和脂肪酸，可举出(6Z,9Z,12Z)-6,9,12-十八碳三烯酸、(8Z,11Z,14Z)-8,11,14-二十碳三烯酸、(5Z,8Z,11Z,14Z)-5,8,11,14-二十碳四烯酸、(4Z,7Z,11Z,14Z,17Z)-4,7,11,14,17-二十二碳五烯酸、(9Z,12Z,15Z)-9,12,15-十八碳三烯酸、(6Z,9Z,12Z,15Z)-6,9,12,15-十八碳四烯酸、(8Z,11Z,14Z,17Z)-8,11,14,17-二十碳四烯酸、(5Z,8Z,11Z,14Z,17Z)-5,8,11,14,17-二十碳五烯酸、(4Z,7Z,10Z,13Z,16Z,19Z)-4,7,10,13,16,19-二十二碳六烯酸、(6Z,9Z)-6,9-十八碳二烯酸、(8Z,11Z)-8,11-二十碳二烯酸、(5Z,8Z,11Z)-5,8,11-二十碳三烯酸等，作为脂质，可举出含上述的不饱和脂肪酸的三酰基甘油酯、二酰基甘油酯、单酰基甘油酯、磷脂酰胆碱、磷脂酰丝氨酸、磷脂酰乙醇胺、磷脂酰肌醇、磷脂酸、溶血磷脂酰胆碱、溶血磷脂酰丝氨酸、溶血磷脂酰乙醇胺、溶血磷脂酰肌醇、溶血磷脂酸、心磷脂或，甾醇等。
作为蛋白质，可举出脂肪酶、磷酸脂肪酶、蛋白酶、转谷氨酰胺酶、淀粉酶、异淀粉酶、葡萄糖异构酶、葡萄糖苷酶、支链淀粉酶、聚半乳糖醛酸酶、甘露糖异构酶、阿拉伯呋喃糖酶、鼠李糖苷酶、甘露糖苷酶、环糊精生成酶、葡萄糖脱氢酶、葡萄糖-6-磷酸脱氢酶、己糖激酶、葡萄糖氧化酶、唾液酸基转移酶、岩藻糖基转移酶、N-乙酰葡萄糖胺基转移酶、半乳糖基转移酶、甘露糖基转移酶、N-乙酰半乳糖胺酶、N-酰基氨基酸消旋酶、D-氨基酰化酶、L-苯基丝氨酸脱氢酶、羰基还原酶、α-酮基酸还原酶、过氧化酶、漆酶、胆甾醇氧化酶、果糖基氨基酸氧化酶、锰过氧化酶、抗坏血酸氧化酶、葡萄糖氧化酶、腈水解酶、腈水合酶、植酸酶、延胡索酸酶、尿酸酶、过氧化氢酶、磷酸酶、脲酶、纤维素酶、转化酶、天冬酰胺酶、鞣酸酶、乳糖酶、尿激酶等，作为肽，可举出谷胱甘肽等。
作为维生素，可举出抗坏血酸、核黄素、硫胺素、烟酸、生物素、氰基钴胺、维生素A、维生素D、维生素K、类胡萝卜素类等，作为糖，可举出葡萄糖胺、N-乙酰葡萄糖胺、木糖、核糖、果糖寡糖、半乳糖寡糖、甘露聚糖寡糖、壳多糖、壳聚糖、透明质酸、粘性多糖类等，作为糖醇，可举出木糖醇、赤藓糖醇、半乳糖醇、甘露糖醇、山梨糖醇等。
作为醇，可举出乙醇、丁醇、甘油等，作为有机酸，可举出醋酸、乳酸、丙酮酸、琥珀酸、柠檬酸、曲酸、富马酸、苹果酸、葡萄糖酸、衣康酸、蚁酸、丙酸、酪酸、异酪酸、丙烯酸、甲基丙烯酸、山梨酸、马来酸、桂皮酸、醛糖酸、酒石酸、异柠檬酸、酮基醛糖酸、2-酮基古洛糖酸等，作为抗生物质，可举出苯并醌系抗生物质、蒽环系抗生物质等。
作为生理活性物质，可举出烟酰胺腺嘌呤二核苷酸、黄素腺嘌呤二核苷酸、乳清酸、莽草酸、叶酸、羟基柠檬酸、脑苷脂、虾青素、S-腺苷基甲硫氨酸、S-甲基甲硫氨酸锍氯化物、类视黄醇类、脑苷脂、辅酶A、辅酶Q10、肌醇、胆碱、肉毒碱、吡咯并喹啉醌、赤霉素、脱落酸、聚羟基链烷酸酯、环孢菌素A、美百乐镇、聚谷氨酸、聚赖氨酸、甘露糖基赤藓糖醇脂质等。
优选而言，作为不饱和脂肪酸，是(6Z,9Z,12Z)-6,9,12-十八碳三烯酸、(8Z,11Z,14Z)-8,11,14-二十碳三烯酸、(5Z,8Z,11Z,14Z)-5,8,11,14-二十碳四烯酸、(4Z,7Z,11Z,14Z,17Z)-4,7,11,14,17-二十二碳五烯酸、(9Z,12Z,15Z)-9,12,15-十八碳三烯酸、(6Z,9Z,12Z,15Z)-6,9,12,15-十八碳四烯酸、(8Z,11Z,14Z,17Z)-8,11,14,17-二十碳四烯酸、(5Z,8Z,11Z,14Z,17Z)-5,8,11,14,17-二十碳五烯酸、(4Z,7Z,10Z,13Z,16Z,19Z)-4,7,10,13,16,19-二十二碳六烯酸、(6Z,9Z)-6,9-十八碳二烯酸、(8Z,11Z)-8,11-二十碳二烯酸、(5Z,8Z,11Z)-5,8,11-二十碳三烯酸等，作为脂质，是含上述的不饱和脂肪酸的三酰基甘油酯、二酰基甘油酯、单酰基甘油酯、磷脂酰胆碱、磷脂酰丝氨酸、磷脂酰乙醇胺、磷脂酰肌醇、磷脂酸、溶血磷脂酰胆碱、溶血磷脂酰丝氨酸、溶血磷脂酰乙醇胺、溶血磷脂酰肌醇、溶血磷脂酸、心磷脂或，甾醇等。
更优选而言，作为不饱和脂肪酸，是(8Z,11Z,14Z)-8,11,14-二十碳三烯酸、(5Z,8Z,11Z,14Z)-5,8,11,14-二十碳四烯酸、(5Z,8Z,11Z)-5,8,11-二十碳三烯酸，作为脂质，是含上述的不饱和脂肪酸的三酰基甘油酯、二酰基甘油酯、单酰基甘油酯、磷脂酰胆碱、磷脂酰丝氨酸、磷脂酰乙醇胺、磷脂酰肌醇、磷脂酸、溶血磷脂酰胆碱、溶血磷脂酰丝氨酸、溶血磷脂酰乙醇胺、溶血磷脂酰肌醇、溶血磷脂酸。
即便现在不是利用微生物制备的物质，可利用微生物制备时，可适应本发明方法。由微生物生产的有用物质是，单独的也可，2种以上的多种的混合物也可。另外，是含上述有用物质作为构成成分的物质也可。
本发明中使用的微生物不特别限制，只要是在由发酵法的有用物质的制备中使用的微生物，可使用任何。另外，即便是至今未在发酵生产中利用的微生物，只要有生产有用物质的能力，可适应本发明方法。另外，即便是原本无生产有用物质的能力的微生物，只要是通过利用作为本领域技术人员所知的方法的突变或重组DNA技术等的育种来赋予生产有用物质的能力的微生物，也可在本发明方法中使用。
作为本发明中使用的具体性的微生物，可举出属于埃希氏菌（Escherichia）属、棒杆菌（Corynebacterium）属、芽孢杆菌（Bacillus）属、沙雷氏菌（Serratia）属、欧文氏菌（Erwinia）属、假单胞菌（Pseudomonas）属、红杆菌（Rhodobacter）属、沙门氏菌（Salmonella）属、弧菌（Vibrio）属、黄单胞菌（Xanthomonas）属、硫还原球菌（Desulfurococcus）属、热球菌（Thermococcus）属、节杆菌（Arthrobacter）属、肠杆菌（Enterobacter）属、醋酸杆菌（Acetobacterium）属、醋杆菌（Acetobacter）属、甲烷丝菌（Methanothrix）属、土壤杆菌（Agrobacterium）属、动胶菌（Zooglea）属、产碱菌（Alcaligenes）属、肠球菌（Enterococcus）属、异常球菌（Deinococcus）属、塞贝菌（Sebekia）属、纤维单胞菌（Cellulomonas）属、隐球菌（Cryptococcus）属、不动杆菌（Acinetobacter）属、土芽孢杆菌（Geobacillus）属、无色嗜盐菌（Natrialba）属、微小杆菌（Exiguobacterium）属、鞘氨醇单胞菌（Sphingomonas）属、热双歧菌（Thermobifida）属、明串球菌（Leuconostoc）属、乳杆菌（Lactobacillus）属、嗜酸菌（Acidiphilium）属、拟诺卡菌（Nocardiopsis）属、链霉菌（Streptomyces）属、无枝酸菌（Amycolata）属、假诺卡菌（Pseudonocardia）属、热单孢菌（Thermomonospora）属、链轮丝菌（Streptoverticillium）属、戈登菌（Gordona）属、拟无枝酸菌（Amycolatopsis）属、火球菌（Pyrococcus）属、热球菌（Thermococcus）属、热原体（Thermoplasma）属、栖热菌（Thermus）属、片球菌（Pediococcus）属、酵母（Saccharomyces）属、结合酵母（Zygosaccharomyces）属、念珠菌（Candida）属、有孢圆酵母（Torulaspora）属、克鲁维酵母（Kluyveromyces）属、毕赤酵母（Pichia）属、假发酵菌（Pseudozyma）属、德巴利酵母（Debaryomyces）属、管囊酵母（Pachysolen）属、被孢霉（Mortierella）属、被孢霉（Mortierella）亚属、耳霉（Conidiobolus）属、腐霉（Pythium）属、疫霉（Phytophthora）属、枝孢菌（Cladosporium）属、红酵母（Rhodotorula）属、虫霉（Entomophthora）属、刺孢子囊菌（Echinosporangium）属、水霉（Saprolegnia）属、吾肯氏壶菌（Ulkenia）属、裂殖壶菌（Schizochytrium）属、破囊壶菌（Thraustochytrium）属、被孢霉（Mortierella）属、木霉（Trichoderma）属、曲霉（Aspergillus）属、根霉（Rhizopus）属、支顶孢菌（Acremonium）属、青霉（Penicillium）属、茎点霉（Phoma）属、漆斑菌（Myrothecium）属、焙烙茸菌属、菌寄生菌（Hypomyces）属、镰孢菌（Fusarium）属、木耳（Auricularia）属、腐霉（Pythium）属、拟月孢菌（Menisporopsis）属、嗜热丝孢菌（Thermomyces）属、尾孢菌（Cercospora）属、葡萄孢菌（Botrytis）属、匍柄霉（Stemphylium）属、红曲霉（Monascus）属、毛霉（Mucor）属、盘多毛孢霉（Pestalotia）属、射脉菌（Phlebia）属、裂褶菌（Schizophyllum）属、绿曲菌（Chloroflexus）属、小圆孢菌（Chaetasbolisia）属、金孢子菌（Chrysosporium）属的微生物等，只要是在有用物质的制备中使用的微生物，不对这些加以限制。
更具体而言，可举出以下的微生物。例如生产的有用物质是不饱和脂肪酸或脂质时，优选被孢霉（Mortierella）属丝状菌，更优选被孢霉（Mortierella）亚属丝状菌，特别优选高山被孢霉（Mortierellaalpina）。例如，作为有含(5Z,8Z,11Z,14Z)-5,8,11,14-二十碳四烯酸作为构成脂肪酸的脂质的生产能力的微生物，可举出属于被孢霉（Mortierella）属、耳霉（Conidiobolus）属、腐霉（Pythium）属、疫霉（Phytophthora）属、青霉（Penicillium）属、枝孢菌（Cladosporium）属、毛霉（Mucor）属、镰孢菌（Fusarium）属、曲霉（Aspergillus）属、红酵母（Rhodotorula）属、虫霉（Entomophthora）属、刺孢子囊菌（Echinosporangium）属、水霉（Saprolegnia）属的微生物。
更具体而言，在属于被孢霉（Mortierella）属被孢霉（Mortierella）亚属的微生物中，可举出例如长形被孢霉（Mortierella elongata）、微小被孢霉（Mortierella exigua）、喜湿被孢霉（Mortierellahygrophila）、高山被孢霉（Mortierella alpina）等。具体而言，可举长形被孢霉（Mortierella elongata）IFO8570、微小被孢霉（Mortierellaexigua）IFO8571、喜湿被孢霉（Mortierella hygrophila）IFO5941、高山被孢霉（Mortierella alpina）IFO8568、ATCC16266、ATCC32221、ATCC32222、ATCC42430、CBS219.35、CBS224.37、CBS250.53、CBS343.66、CBS527.72、CBS529.72、CBS608.70、CBS754.68等的菌株。
这些的菌株均可从独立行政法人制品评价技术基盘机构生物遗传资源部门（NBRC）、及美国的美国典型培养物保藏中心（AmericanType Culture Collection，ATCC）及，Centrral Bureau voorSchimmelcultures（CBS）等得到。另外，含有(5Z,8Z,11Z,14Z)-5,8,11,14-二十碳四烯酸或(5Z,8Z,11Z,14Z)-5,8,11,14-二十碳四烯酸的脂质的生产的情况中，可使用本发明人从土壤分离的菌株高山被孢霉（Mortierella alpina）1S-4株、菌株长形被孢霉（Mortierella elongata）SAM0219（微工研菌寄第8703号）（微工研条寄第1239号）。含有(8Z,11Z,14Z)-8,11,14-二十碳三烯酸或(8Z,11Z,14Z)-8,11,14-二十碳三烯酸的脂质的生产的情况中，可使用从1S-4株由作为常规方法的亚硝基胍变异法诱导得到的，是Δ5不饱和化活性缺失的变异株的高山被孢霉（Mortierella alpina）SAM1860（微工研条寄第3589号、FERM BP-3589）、高山被孢霉（Mortierellaalpina）S14株、高山被孢霉（Mortierella alpina）Iz3株等。含有(5Z,8Z,11Z)-5,8,11-二十碳三烯酸或(5Z,8Z,11Z)-5,8,11-二十碳三烯酸的脂质的生产的情况中，作为从1S-4株由作为常规方法的亚硝基胍变异法诱导得到的，Δ12不饱和化活性缺失的变异株，可使用高山被孢霉（Mortierella alpina）SAM1861（微工研条寄第3590号、FERMBP-3590）、高山被孢霉（Mortierella alpina）M209-7株（专利生物保藏中心保藏编号FERM P-15766）、高山被孢霉（Mortierella alpina）JT180株。
微生物的预培养是用适宜于使用的微生物的培养基，向主培养在得到必要的菌体数的条件下进行。
得到的预培养液的向主培养初始培养基的接种可由本领域技术人员根据培养方法适宜选择。具体而言，将微生物菌株的营养细胞、胞子和/或菌丝、或者预先培养而得到的种培养液或由种培养回收的营养细胞、胞子和/或菌丝接种到液体培养基或固体培养基而培养。
在主培养基，作为碳水化物或烃的氧化物，只要是将葡萄糖、果糖、木糖、蔗糖（蔗糖）、麦芽糖、可溶性淀粉、糖蜜、甘油、甘露糖醇、山梨糖醇、半乳糖、糖化淀粉等单独或组合的，还有含有这些的柠檬糖蜜、甜菜糖蜜、甜菜榨汁、甘蔗榨汁等、培养的微生物可作为碳源资用的，或者本领域技术人员一般地使用的，就不限制，任何均可使用。初始的碳水化物和烃的氧化物的合计浓度只要以碳当量计不足0.4重量%即可，优选为0.2重量%以下，更优选为0.1重量%以下，再者优选为0.05重量%以下，特别是优选为0重量%、即实质上初始培养基中未添加碳水化物和/或烃的氧化物是好的。其中，以碳当量计0.4重量%是指，以葡萄糖计为1重量%、以蔗糖计为0.95重量%、以乙醇计为0.77重量%、以甘油计为1.02重量%。自预培养的微量的碳水化物和/或烃的氧化物的具有通常是微量，对应于本发明中所述“主培养初始培养基实质上未添加碳水化物和/或烃的氧化物”。另外，即便添加有不预定被微生物代谢利用的，不成碳源的碳水化物及/烃的氧化物，也对应于“主培养初始培养基实质上未添加碳水化物和/或烃的氧化物”。
将预培养液接种到主培养初始培养基中后，优选为微生物的适应期（Lag Phase）结束后向对数增殖期（Log Phase）移行之后，将碳水化物和/或烃的氧化物添加到培养基中。微生物的适应期、对数增殖期根据有用物质的发酵生产中使用的微生物、培养基、培养条件等而不同，由本领域技术人员公知的方法测定细胞数、培养液的浊度、每培养液的干燥或湿菌体量等，另外，测定培养液的pH或溶解氧浓度等或，从培养装置排出的气体中的二氧化碳浓度等，可通过确认各参数的推移来确认微生物的适应期、对数增殖期。例如在以细胞数作为参数使用时，细胞数开始指数函数地增加的时期成从适应期移行到对数增殖期的时期。另外，用溶解氧浓度研究时，消费氧开始指数函数地增加的时期成从适应期移行到对数增殖期的时期。
将碳水化物和/或烃的氧化物流加的次数只要是1次以上，多次也可，另外，可连续或间歇地将碳水化物和/或烃的氧化物添加到培养基。添加的碳水化物和/或烃的氧化物的总量不特别限制。
作为氮源，可单独或多种，或者组合使用以下氮源：除了微生物培养中一般地使用的，例如，胨、酵母提取物、麦芽提取物、肉提取物、酪蛋白氨基酸、玉米浆、大豆蛋白、脱脂大豆、棉籽滓、酪蛋白水解物、各种发酵菌体、及其消化物等的天然氮源之外，还有尿素等的有机氮源、以及硝酸钠、硝酸铵等的硝酸盐、硫酸铵、氯化铵、磷酸铵等的无机酸或有机酸的铵盐、氨等的无机氮源。
此外根据需要，可将磷酸根离子、钾离子、钠离子、镁离子、钙离子或，铁、铜、锌、锰、镍、钴等的金属离子或，生物素、硫胺素等的维生素等作为微量营养源使用。根据需要，可添加アデカネート、硅酮等的消泡剂。
主培养可使用既有的培养微生物的方法、具体而言固体培养、静置培养、振荡培养、通气搅拌培养等。作为培养操作法，可使用所谓的回分培养、流加回分培养、反复回分培养及连续培养等之任何。再有，作为搅拌手段，可使用例如叶轮（搅拌叶轮）、气升式发酵槽、发酵肉汤的泵驱动循环、或者这些手段的组合等。
本发明的制备方法可由上述的培养方法实施，但在其中也是，在液体培养基中用通气搅拌培养进行主培养在工业上有利。作为用于进行通气搅拌培养的槽，可使用例如，发酵缸及箱等的搅拌槽，但不限于这些。
在通气搅拌培养中，将例如空气等的含有氧的气体或，氩及氮等的不含有氧的气体之任何通气都可，另外，将那些气体混合通气也可，这样的气体根据培养系统的条件适宜选择即可。
培养温度随着所使用的微生物而不同，通常、5～100℃、优选为10～50℃、更优选为15～40℃。作为另外一实施方式，在对于微生物的培育适宜的温度培养而增殖微生物后，在适宜于目的物质的生产的温度持续培养，提高目的物质的生产性也可。
作为预培养在固体培养基或，试管或瓶中的液体培养基培养的时，培养期间通常是5小时～10天、优选为5小时～5天、更优选是5小时～3天。持续生产有用物质的主培养的培养期间通常是5小时～30天、优选为5小时～20天、更优选是5小时～15天。可将所望的有用物质的生产量或培养基中的营养成分、底物成分的残留量作为主培养结束时的判断基准。
再有，培养微生物的培养基的pH是3～9即可，可使用无机或有机的酸、碱溶液、尿素、碳酸钙、氨等调节pH。
如此培养微生物而在微生物菌体内、菌体表面、或者培养基中蓄积目的物质。将含微生物菌体的培养液本身或其浓缩物作为最终的目的物质也可，但也可根据需要采集有用物质、纯化。目的有用物质在微生物菌体内或菌体表面蓄积时，可通过离心分离、过滤等的通常的方法从培养液回收微生物菌体，将微生物菌体本身作为最终的目的物质，另外，可用溶剂等回收目的物质，使用离子交换层析、凝胶过滤层析、吸附层析、薄层层析、高效液相层析、晶析等、通常的目的物质的纯化方法，纯化目的物质。另外，目的物质在培养基中蓄积时，也可将分离微生物菌体的培养液本身或其浓缩物作为最终的目的物质，但使用上述的通常的纯化法纯化目的物质也可。
作为生产是有用物质的不饱和脂肪酸或脂质的微生物，可举属于被孢霉（Mortierella）属、耳霉（Conidiobolus）属、腐霉（Pythium）属、疫霉（Phytophthora）属、青霉（Penicillium）属、枝孢菌（Cladosporium）属、毛霉（Mucor）属、镰孢菌（Fusarium）属、曲霉（Aspergillus）属、红酵母（Rhodotorula）属、虫霉（Entomophthora）属、刺孢子囊菌（Echinosporangium）属、水霉（Saprolegnia）属的微生物，这些的微生物在微生物菌体内蓄积是有用物质的不饱和脂肪酸或脂质。
特别是，由于将碳水化物和/或烃的氧化物由外型淀粉酶异化的微生物分解以淀粉为代表的高分子量α-聚糖的活性低，通过代谢异化碳水化物和/或烃的氧化物的速度变慢，会成为主培养初始培养基中实质上不含碳水化物和/或烃的氧化物的状态，由此优选。
将这些的碳水化物和/或烃的氧化物由外型淀粉酶异化的微生物，因为在微生物开始对数增殖之后，添加的碳水化物和/或烃的氧化物是糖类时，可与添加的糖类立即反应，所以是特别优选的。
培养结束后，由本领域技术人员熟知的方法，例如参照特开2000-69987等的方法，可从培养基得到不饱和脂肪酸、含有不饱和脂肪酸的脂质、或者含有这些的微生物菌体。
微生物菌体，如所望，在杀菌后，优选干燥。干燥可通过烘箱加热、冷冻干燥、热风干燥等进行。从干燥菌体或湿菌体，使用本领域技术人员熟知的方法可得含有不饱和脂肪酸的脂质。例如，将干燥菌体用己烷等的有机溶剂提取处理后，通过在减压下从提取物除去有机溶剂，得到以三甘油酯为主的，含有高浓度的不饱和脂肪酸的脂质。另外，通过向得到的以三甘油酯为主的脂质进行脱树胶、脱酸、脱色、除臭等，对通常的食用油脂实施的纯化处理，可得高纯度的纯化食用油脂（三甘油酯）。
脂质中的不饱和脂肪酸可直接分离，可通过作为与低级醇的酯、例如甲基酯，从其他脂质成分容易地分离。另外，可将仅所望的不饱和脂肪酸从其他不饱和脂肪酸容易地分离。这样的分离方法为本领域技术人员所熟知。
本发明的微生物菌体可直接，或者干燥而利用。另外，用本发明的制备方法得到的不饱和脂肪酸及含有其的脂质也可以各种各样的方法利用。这些可向，例如，辅助物、营养组合物、动物用饲料（特别是宠物食品）、水产类养殖用饲料、含乳粉等的食品，使用本领域技术人员熟知的方法而利用。
接下来示实施例，但本发明不限于下述的实施例。
【实施例】
【实施例1】
【<2>由向初始培养基添加葡萄糖的培养系统的(5Z,8Z,11Z,14Z)-5,8,11,14-二十碳四烯酸的生产】
使用高山被孢霉（Mortierella alpina）1S-4株。将冷冻孢子液接种到100L、酵母提取物1.0w/w%、含水葡萄糖2.0w/w%、pH6.3的培养基100mL中，在往复振荡100rpm、温度28℃的条件下开始预培养（第一阶段），培养3天。接下来，将酵母提取物1.0w/w%、含水葡萄糖2.0w/w%、大豆油0.1w/w%、pH6.3的培养基30L在50L容培养槽中制备，向其接种200mL种培养液（第一阶段），开始预培养（第二阶段），在温度28℃的条件下培养2天。接下来，向主培养的初始培养基（2.67w/w%含水葡萄糖、5.0w/w%脱脂大豆粉、0.3w/w%K2HPO4、0.05w/w%MgCl2．6H2O、0.05w/w%CaCl2．2H2O、pH6.0）接种25L种培养液（第二阶段），合计4000L的初始培养液量。
于温度26℃、内压0.10MPa开始培养。进行培养第1天为5.33%、培养第2天为5.33%、培养第3天为4.00%、培养第4天为4.00%、第5天为2.67%的含水葡萄糖的流加，添加合计24.0%的含水葡萄糖的结果，培养基中的葡萄糖完全地枯渴是培养第12天。在培养第12天(5Z,8Z,11Z,14Z)-5,8,11,14-二十碳四烯酸的生产量是17.7g/L、总脂肪酸中占的(5Z,8Z,11Z,14Z)-5,8,11,14-二十碳四烯酸的比例是42.4%。
【<2>由未向初始培养基添加葡萄糖的培养系统的(5Z,8Z,11Z,14Z)-5,8,11,14-二十碳四烯酸的生产】
使用高山被孢霉（Mortierella alpina）1S-4株。将冷冻孢子液接种到100μL、酵母提取物1.0w/w%、含水葡萄糖2.0w/w%、pH6.3的培养基100mL中，在往复振荡100rpm、温度28℃的条件下开始预培养（第一阶段），培养3天。接下来，将酵母提取物1.0w/w%、含水葡萄糖2.0w/w%、大豆油0.1w/w%、pH6.3的培养基30L在50L容培养槽中制备，向其接种200mL种培养液（第一阶段），开始预培养（第二阶段），在温度28度的条件下培养2天。接下来，向主培养的初始培养基（5.0w/w%脱脂大豆粉、0.3w/w%K2HPO4、0.05w/w%MgCl2．6H2O、0.05w/w%CaCl2．2H2O、pH6.0）接种25L种培养液（第二阶段），合计4000L的初始培养液量。
于温度26℃、内压0.10MPa开始培养。进行培养第1天为5.33%、培养第2天为5.33%、培养3天为5.33%、培养第4天为4.00%、第5天为4.00%的含水葡萄糖的流加，与<1>同样地添加合计24.0%的含水葡萄糖的结果，培养基中的葡萄糖完全地枯渴是培养第9天。在培养第12天(5Z,8Z,11Z,14Z)-5,8,11,14-二十碳四烯酸的生产量是18.3g/L、总脂肪酸中占的(5Z,8Z,11Z,14Z)-5,8,11,14-二十碳四烯酸的比例是45.6%。在<1>初始葡萄糖添加培养和<2>初始葡萄糖未添加培养中，培养基中的葡萄糖浓度的推移显示于图1，总脂肪酸中占的(5Z,8Z,11Z,14Z)-5,8,11,14-二十碳四烯酸的比例的推移显示于图2。
【实施例2】
【<1>由向初始培养基添加葡萄糖的培养的(8Z,11Z,14Z)-8,11,14-二十碳三烯酸的生产】
使用高山被孢霉（Mortierella alpina）S14株。将高山被孢霉（Mortierella alpina）S14株1铂耳左右接种到酵母提取物1.0w/w%、含水葡萄糖2.0w/w%、pH6.3的培养基100mL，在往复振荡100rpm、温度28℃的条件下开始预培养（第一阶段），培养3天。接下来，将酵母提取物1.0w/w%、含水葡萄糖2.0w/w%、大豆油0.1w/w%、pH6.3的培养基30L在50L容培养槽中制备，向其接种200mL种培养液（第一阶段），开始预培养（第二阶段），在温度28℃的条件下培养2天。接下来，向主培养的初始培养基（2.00w/w%含水葡萄糖、4.32w/w%脱脂大豆粉、0.3w/w%K2HPO4、0.05w/w%MgSO4．7H2O、0.05w/w%CaSO4．2H2O、pH6.3）接种25L种培养液（第二阶段），合计4000L的初始培养液量。
于温度26℃、内压0.10MPa开始培养。进行培养第1天为4.00%、培养第2天为5.00%、培养第3天为6.00%、培养第6天为6.00%的含水葡萄糖的流加，添加合计23.0%的含水葡萄糖，再在培养第4天添加1.00%的琥珀酸2钠、0.021%的NaOH的结果，在培养第13天培养基中的残留葡萄糖浓度是0.75%。
【<2>由未向初始培养基添加葡萄糖的培养系统的(8Z,11Z,14Z)-8,11,14-二十碳三烯酸的生产】
使用高山被孢霉（Mortierella alpina）S14株。将高山被孢霉（Mortierella alpina）S14株1铂耳左右接种到酵母提取物1.0w/w%、含水葡萄糖2.0w/w%、pH6.3的培养基100mL，在往复振荡100rpm、温度28℃的条件下开始预培养（第一阶段），培养3天。接下来，将酵母提取物1.0w/w%、含水葡萄糖2.0w/w%、大豆油0.1w/w%、pH6.3的培养基30L在50L容培养槽中制备，向其接种200mL种培养液（第一阶段），开始预培养（第二阶段），在温度28℃的条件下培养2天。接下来，向主培养的初始培养基（4.32w/w%脱脂大豆粉、0.3w/w%K2HPO4、0.05w/w%MgSO4．7H2O、0.05w/w%CaSO4．2H2O、pH6.3）接种25L种培养液（第二阶段），合计4000L的初始培养液量。
在温度26℃、内压0.10MPa开始培养。进行培养第1天为6.00%、培养第2天为5.00%、培养第3天为6.00%、培养第6天为6.00%的含水葡萄糖的流加，添加合计23.0%的含水葡萄糖，再在培养第4天添加1.00%的琥珀酸2钠、0.021%的NaOH，培养基中的葡萄糖完全地枯渴是培养第12天。
【实施例3】
【<1>由向初始培养基添加葡萄糖的培养的(5Z,8Z,11Z)-5,8,11-二十碳三烯酸的生产】
使用高山被孢霉（Mortierella alpina）JT180株。将高山被孢霉（Mortierella alpina）JT180株1铂耳左右接种到酵母提取物1.0w/w%、含水葡萄糖2.0w/w%、pH6.0的培养基20mL，往复振荡100rpm、在温度℃28的条件下开始预培养，培养3天。接下来，向主培养的初始培养基（2.00w/w%葡萄糖、1.00w/w%酵母提取物、pH6.0）接种预培养液20mL，合计5L的初始培养液量。于28℃温度培养2天之后，将培养温度变更到19℃，继续培养。进行培养第1天、培养第2天为各自1.00%的葡萄糖的流加，添加合计4.0%的葡萄糖而进行培养的结果，培养基中的葡萄糖完全地枯渴是培养第6天。
【<2>由未向初始培养基添加葡萄糖的培养系统的(5Z,8Z,11Z)-5,8,11-二十碳三烯酸的生产】
使用高山被孢霉（Mortierella alpina）JT180株。将高山被孢霉（Mortierella alpina）JT180株1铂耳左右接种到酵母提取物1.0w/w%、含水葡萄糖2.0w/w%、pH6.0的培养基20mL，往复振荡100rpm、在温度℃28的条件下开始预培养，培养3天。接下来，向主培养的初始培养基（2.00w/w%葡萄糖、1.00w/w%酵母提取物、pH6.0）接种预培养液20mL，合计5L的初始培养液量。于28℃温度培养2天之后，将培养温度变更到19℃，继续培养。进行培养第1天、培养第2天为各自1.00%的葡萄糖的流加，添加合计4.0%的葡萄糖而进行培养，则培养基中的葡萄糖完全地枯渴是培养第5天。
由以上的结果知，可通过不向初始培养基添加碳水化物和/或烃的氧化物，培养微生物，升高微生物消费碳水化物和/或烃的氧化物的速度，缩短生产有用物质所要求的培养期间，升高有用物质的生产性。
Claims (6)
1.由微生物培养制备有用物质的方法，其包括：
微生物的预培养步骤、
将得到的预培养液接种到主培养初始培养基的步骤，其中在主培养初始培养基中的碳水化物和烃的氧化物的合计浓度以碳当量计不足0.4重量%，
微生物开始对数增殖之后，添加碳水化物和/或烃的氧化物的主培养步骤。
2.权利要求1所述的制备方法，其中主培养初始培养基实质上未添加碳水化物和/或烃的氧化物。
3.权利要求1或2所述的制备方法，其中微生物开始对数增殖之后添加的碳水化物和/或烃的氧化物是糖类。
4.权利要求1～3之任一项所述的制备方法，其中微生物是将碳水化物和/或烃的氧化物由外型淀粉酶异化的微生物。
5.权利要求1～4之任一项所述的制备方法，其中微生物是被孢霉（Mortierella）属微生物。
6.权利要求1～5之任一项所述的制备方法，其中有用物质是高度不饱和脂肪酸。
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| JP6961227B2 (ja) * | 2018-01-17 | 2021-11-05 | 国立研究開発法人産業技術総合研究所 | 抗酸化物質の製造方法 |
| KR102174589B1 (ko) * | 2018-08-03 | 2020-11-05 | 부경대학교 산학협력단 | 뱀장어 사료내 항생제 대체를 위한 신바이오틱스 개발 |
| CN108624638B (zh) * | 2018-08-24 | 2021-03-30 | 湖南汇升生物科技有限公司 | 一种发酵生产氨基葡萄糖的方法 |
| EP3877535A1 (de) * | 2018-11-09 | 2021-09-15 | Evonik Operations GmbH | Verfahren zur herstellung einer leicht aufschliessbaren biomasse mit erhöhtem gehalt an polyungesättigten fettsäuren |
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